The Na,K-ATPase, or sodium pump, generates the ion gradients responsible for most fluid and electrolyte transport processes in the kidney. In keeping with this central role in homeostasis, the function of the renal sodium pump is regulated by signals that contribute to the control of body fluid volume and blood pressure. To drive vectorial trans-epithelial transport, the Na,K-ATPase must be restricted to the basolateral surfaces of renal tubule epithelial cells. While a great deal has been learned about the structure and catalytic cycle of the sodium pump, much less is known about the partner proteins and trafficking pathways that determine its subcellular distribution and modulate its activity. During the previous 4 year funding period of this award we have identified new sodium pump interacting proteins and explored their roles in controlling its properties. We have also adapted a novel labeling methodology that allows us to investigate the attributes of temporally defined cohorts of Na,K-ATPase. The SNAP tag technique endows a protein of interest with the ability to become covalently coupled to fluorophores or biochemically useful ligands. We have generated a SNAP-tagged Na,KATPase a-subunit fusion protein and expressed it in MDCK renal epithelial cells. The SNAP tagged a-subunit assembles with Na,K-ATPase -subunit to form functionally competent pumps that are sorted appropriately. We have developed pulse-chase protocols with which we can observe directly the trafficking itinerary pursued by newly synthesized Na,K-ATPase and isolate newly synthesized Na,K-ATPase in association with its collections of partner proteins. These efforts have already yielded surprising new insights into the mechanisms through which the pump is assembled and transported through epithelial cells. We will expand our analysis to: 1) define the pathways and parameters that govern the trafficking of newly synthesized Na,K-ATPase in polarized renal epithelial cells; 2) identify and characterize the protein partners that interact with a temporally-defined cohort of Na,K-ATPase at each stage of its post-synthetic life span; and 3) establish the pathways and partners that regulate Na,K-ATPase sorting and function in the epithelial cells of each of the segments of the developing kidney and mature nephron in situ. This approach permits us to ask entirely new and previously experimentally inaccessible questions about the biology of the sodium pump, and will yield valuable insights into the cell biological and biochemical factors that govern this physiologically critical ion transport system.